Aims: Our long term objectives are to determine fundamental mechanisms controlling lung endothelial permeability in order to understand the mechanisms of lung injury. Here, we will test the hypothesis that the terminal complement products - SC5b-9 and the Membrane Attack Complex (MAC) - affect lung endothelial barrier properties through cellular mechanisms. Since SC5b-9 contains vitronectin (VN), we will determine the importance of VN-integrin interactions and VN conformational changes in barrier regulation. To quantify barrier properties, we will use our novel intravital technique in single lung microvessels. The extent and time course of barrier responses will be defined before and after exposing single lung arterioles and venules to SC5b-9 and MAC enriched sera which will be cell free, and in which VN will be conformationally modified or unmodified. To interpret mechanisms, barrier responses will be determined in the presence of agents which affect endothelial signal transduction and of the RGD tripeptide which inhibits VN-integrin binding. Procedures: The protocols are planned in isolated blood perfused rat lungs held at constant inflation (alveolar pressure = 5 cmH2O). Endothelial liquid flux measurements will be obtained with our newly developed split drop technique for lung. Subpleural arterioles and venules (identified by pattern of flow distribution) will be continuously viewed and videorecorded by stereomicroscopy. With stopped blood flow at known microvascular pressure (Pmv), the selected microvessel will be microinfused for a determined period with either experimental or control sera. With subsequent micropunctures, an oil drop will be first microinjected into the microvessel, then the drop will be split across a determined length by microinjecting control or experimental sera (split solution). Transendothelial flux (Jv) of the split solution will be interpreted from timed measurements of split length and microvessel diameter. Two to three determinations of Jv at different Pmv will be plotted to obtain the Jv-Pmv relationship. The slope of the Jv-Pmv line will be interpreted as the endothelial hydraulic conductivity (Lp). to estimate the endothelial reflection coefficient (sigma), two Jv-Pmv lines will be determined each with a split solution of a different oncotic pressure, sigma will be interpreted as the ratio of the differences in x-intercept and oncotic pressure between the two lines. Significance: The effects of terminal complement products and plasma VN on lung endothelial barrier properties have not been previously quantified. Therefore these determinations will be the first to define the importance of these products in regulation of lung endothelial permeability. Since terminal products and VN are involved in tissue injury and inflammation, the data will improve our fundamental understanding of lung injury mechanisms, and of cellular mechanisms in endothelial barrier regulation.